1. Field of the Invention
A new class of nucleoside triphosphates that contain an oligonucleotide of random or defined sequence attached to the base and are substrates for polymerases and terminal nucleotidyl transferases is described. The present invention further describes new reactive bifunctional linker amidites that can be used for the synthesis of the new class of nucleoside triphosphates. Furthermore, this invention describes the methods of making these amidites and triphosphates as well as methods of using these nucleoside triphosphates for genetic analysis.
2. Description of Related Art
Sequencing of human and other genomes has provided an array of opportunities in genetic analysis for the purposes of improving human and animal health, food and water safety, forensics, crop protection, etc., to name a few. Genotyping of individuals and populations will allow the feasibility of tailored therapies suited to individuals or patient groups. Identifying metabolic differences in patient groups based upon genetic polymorphism will provide improved dosing regimens, enhancing drug efficacy and safety. Understanding the genetic basis of disease will provide new drug targets, decrease drug development costs, increase chances of success in clinical development and overall maximize commercial potential. Similarly understanding the genetic basis of disease in animal and plants will help engineer disease resistant animals & crops as well as enhance desirable characteristics.
In addition to looking at genetic differences, differential expression analysis of various genes is very important. In many cases, genes relevant for a particular disease may be expressed in very low copy number. Identifying and quantitating these low copy genes requires very high sensitivity assays and will generally require some type of amplification. A number of amplification methods, such as Ligase Chain reaction (LCR), Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Self-Sustained Sequence Replication (3SR) also known as Nucleic Acid Sequence Based Amplification (NASBA), QB Replicase amplification and Rolling Circle Amplification (RCA), have been developed (J. Virological Methods, 1991, 35, 117–126). A generally accepted definition of amplification and the term amplification, as referred to herein, includes making copies of an original sequence and/or a complementary sequence. Most of these methods have high background as sequences being amplified although unique are not substantially different from sequences in other regions of the genome. Therefore a method that adds a highly unique sequence to the target for amplification and/or detection can provide higher sensitivity and hence is desirable.
Fluorescence detection methods are widely used for detection of DNA or RNA due to their high sensitivity and lower toxicity compared to radiolabels. For a number of applications and especially for multiplexing, several fluorophores with distinguishable characteristics are required. For example, in sequencing, 4 different fluorescent dyes are used to distinguish different nucleoside bases. An ideal set of dyes will absorb at the same wavelength, but emit at readily differentiable wavelengths at similar intensities. Unfortunately, it is extremely difficult to find such a set of dyes. In order to circumvent this problem, fluorescence energy transfer systems have been designed, where a single dye is used to absorb energy which then acts as a donor to transfer energy to a set of acceptor dyes emitting at different wavelengths. For genetic analysis, including sequencing, energy transfer systems have been used in two ways; primers labeled with energy transfer dyes (Ju. J. et. al., Proc. Natl. Acad. Sci. USA 1995, 92, 4347–4351; Hung S. C. et. al., Anal. Biochem. 1996, 243, 15–27 and Hung S. C. et. al., Anal. Biochem. 1997, 252, 78–88) and nucleoside triphosphates labeled with energy transfer dyes (Rosenblum B. B. et. al., Nucleic Acids Res., 1997, 25, 4500–4504; Lee L. G. et. al., Nucleic Acids Res. 1997, 25, 2816–2822; Nampalli, S. et. al. Tet. Lett. 2000, 41, 8867–8871; Lee L. G. et. al., 1999, U.S. Pat. No. 5,863,727; Kumar S, et. al. 2000, PCT WO 00/13026; Rao, T. S. et. al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20, 673–676). The latter is desirable as it is easier to separate labeled nucleoside triphosphates from labeled product than labeled product from labeled primer. In addition labeled primers also result in higher background as all extension products are labeled.
However, energy transfer between dyes attached to different bases on a primer has been found to be more efficient than when the dyes are attached to each other via other linkers. The reasons for this are not well understood, but it is possible that the right conformation with the linkers tried so far, has not been achieved or the DNA bases somehow facilitate the energy transfer. Therefore it is desirable to combine the desirable properties of both systems.
Accordingly, there is a need for high sensitivity, high specificity methods that are easily multiplexed. This concern is addressed in greater detail below.